(1) Field of the Invention
The present invention relates to a novel polypeptide, also referred to as a bacteriocin, produced by Lactococcus lactis subspecies lactis which is inhibitory against selected Gram-positive bacteria, to a method of inhibiting the Gram-positive bacteria with the bacteriocin and to methods for producing the bacteriocin. In particular, the present invention relates to a bacteriocin encoded by DNA from Lactococcus lactis subspecies lactis NRRL-B-18809 and to a precursor polypeptide which is post translationally modified after expression of the DNA.
(2) Prior Art
Lactic acid bacteria comprise a group of Gram-positive spherical and rod-shaped bacteria that are usually non-pathogenic and fermentative. The genera included in this group are Lactococcus, Streptococcus, Lactobacillus and Pediococcus. Many members of these genera are traditionally used in food fermentations and are considered "generally regarded as safe" (GRAS). Production of antagonistic substances and bacteriocins by lactic acid bacteria has been widely reported (Klaenhammer, T. R., Biochimie. 70:337-349 (1988)). Some of the bacteriocins elaborated by these bacteria have a rather narrow spectrum (for example, closely related species) while others exhibit a broader spectrum crossing even the genus grouping. Members of the genus Lactococcus commonly used as starter cultures in fermented foods are Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. cremoris. Both these species produce bacteriocins. A well characterized and nomenclaturally recognized bacteriocin from Lactococcus lactis subsp. lactis is nisin. Lactococcus lactis subsp. cremoris produces diplococcin. Nisin has a wide antibacterial spectrum (Hurst, A., Advances Appl. Microbiol. 27:85-123 (1981)) while diplococcin is primarily active against Lactococcus lactis subsp. lactis and other non-bacteriocin producing Lactococcus lactis subsp. cremoris strains (Foster, E. M., F. E. Nelson, M. L. Speck, R. N. Doetsch, and J. C. Olson, Jr., Dairy Microbiology, Prentice-Hall Inc., Englewood Cliffs, N.J. p.15 (1957)). Because of the wide antibacterial activity of nisin and its potential applications (Hurst, A., Advances Appl. Microbiol. 27:85-123 (1981); Delves-Broughton, J. Food Technology, 44:100-112, 117 (1990)), a search for other strains within this group that elaborate different bacteriocin(s) with wide antibacterial spectra was made.
Structural characteristics of nisin are well documented in several review articles (Klaenhammer, T. R., Biochimie, 70:337-349 (1988); Geis, A., Kieler Milchwirtschaftliche Forschungsberichte 41:97-104 (1989)). The nucleotide sequence of the nisin gene is known (Buchman, G. W., et al., J. Biol. Chem. 263:16260-16266 (1988)) and descriptions of its post-translational modifications (Kaletta, C. and K-D Entian., J. Bacteriol. 171:1597-1601 (1989)) and degradation products (Chan, W. C., et al., FEBS Lett. 252:29-36 (1989); Barber, M., et al., Experentia 44:266-270 (1988)) have appeared. The solution conformation of nisin was determined (Slijper, M., et al., FEBS Lett. 252:22-28 (1989)) as well as other physical and chemical properties (Liu, W. and J. N. Hansen, Appl. Environ. Microbiol. 56:2551-2558 (1990)).
Nisin is similar to other known lanthionine containing bacteriocins, including epidermin (Schnell, N., et al., Nature 333:276-278 (1988)), pep 5 (Weil, H-P., et al., Eur. J. Biochem. 194:217-223 (1990)), lanthiopeptin (Wakamiya, T., et al., Tet. Lett. 29:4771-4772 (1988)), ancovenin (Wakamiya, T., et al., Tet. Lett. 29:4771-4772 (1988)), subtilin (Buchman, G. W., et al., J. Biol. Chem. 263:16260-16266 (1988)), and gallidermin (Kellner, R. and G. Jung., Proc. 20th Europ. Peptide Sym. 366-368 (1988)). The aforementioned lantibiotics share characteristics of small (3500 Da), basic (pI&gt;10) post-translationally modified proteins with an inhibitory spectrum limited to gram positive bacteria. Nisin has been attributed the ability to limit sporulation of Bacilli and Clostridia.
U.S. Pat. No. 4,883,673 to Gonzalez describes a pediocin. U.S. Pat. No. 4,929,445 to Vandenbergh et al describes the DNA encoding the pediocin. U.S. Pat. Nos. 4,740,593 and 4,716,115 to Gonzalez et al describe microorganisms containing DNA from a nisin producing microorganism transferred to a non-nisin producing microorganism using various techniques for manipulating the DNA. The prior art also describes the production of antifungal substances from Lactococcus and Pediococcus which are unrelated to the present invention.
U.S. patent application Ser. No. 07/492,969 assigned to a common assignee describes bacteriocin LL-1 from Lactococcus lactis NRRL-B-18535. This strain produces a bacteriocin which is related to nisin whereas the bacteriocin LL-2 of the present invention is different from nisin.